Single-Cell Genetic Analysis Using Automated Microfluidics to Resolve Somatic Mosaicism

Szulwach, Keith E.; Chen, Peilin; Wang, Xiaohui; Wang, Jing; Weaver, L. Suzanne; Gonzales, Michael L.; Sun, Gang; Unger, Marc; Ramakrishnan, Ramesh

doi:10.1371/journal.pone.0135007

Cited by 42 publications

(58 citation statements)

References 32 publications

(39 reference statements)

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“…For a single-cell coverage depth ≥15 and a mutationSeq SNV-calling probability threshold of 90%, the FPR and TPR were 9.66 × 10 −6 and 68.8%, respectively. This FPR is comparable to that obtained by commercial microfluidic MDA using whole exome sequencing data from a cell in unknown cell phase (22).…”

Section: Resultssupporting

confidence: 67%

“…We assessed the bias of droplet MDA relative to the following published methods that have been shown to achieve high coverage breadth: multiple annealing and looping-based amplification cycles (MALBAC) on single cancer cell line cells (15), MDA of single sperm cells in custom microfluidic devices (referred to herein as "custom microfluidic MDA") (7), microwell MDA of single neurons (MIDAS) (17), MDA of single cancer cell line cells in G2/M phase (nuc-seq) (4), WGA of genomic fragments partitioned into picoliter droplets performed on single endothelial cells (eWGA) (21), and MDA of single B-lymphoblast cell line cells in a commercially available microfluidic device (referred to herein as "commercial microfluidic MDA") (22). We analyzed all publicly available single-cell WGS data in each study, using data from normal diploid cells for comparison wherever possible.…”

Section: Resultsmentioning

confidence: 99%

“…This approach preserves the programmable and multistep nanoliter-volume processing of our previously reported microfluidic droplet-based device (13), but without the need for any specialized microfabrication. In contrast to closed-format microfluidic devices that require multiple sizes of trapping structures to isolate single cells of various diameters (22), the droplet MDA method is able to isolate cells of different sizes without any modification. Unlike similar systems that formulate nanoliter-volume single-cell reactions on an open array (17,18), droplet MDA avoids the possibility of crosscontamination between reactions by using noncontact dispensing to place reactions in spatially distinct locations separated by oil.…”

Section: Discussionmentioning

confidence: 99%

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Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

Leung

Klaus

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in highthroughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform highthroughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10 −6 and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.single-cell sequencing | whole genome amplification | multiple displacement amplification | microdroplet | nanoliter volume

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

Leung

Klaus

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Recent advances in genomics, especially in single-cell genomics, have revealed that genome mosaicism in human somatic tissues is a far more frequent phenomenon than once believed (22)(23)(24)(25)(26). In this study, we demonstrated a great degree of variation in genome mosaicism among different somatic tissue types.…”

Section: Discussionsupporting

confidence: 48%

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations

Kwon

Connelly

Hansen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.iPSCs | fibroblasts | reprogramming | genomic variation | exome sequencing G enomic integrity of human induced pluripotent stem cells (iPSCs) is an unresolved question and a crucial issue for iPSCs-based therapeutics. To understand the scope of acquired genetic changes that occur during iPSC generation, previous studies have compared single nucleotide variants (SNVs), copy number variations (CNVs), and chromosomal rearrangements in iPSCs to donor somatic cells or embryonic stem cells (ESCs) using various assays, including SNP array and next-generation sequencing methods (1-6). Whole genome or exome sequencing (WGS/ WES) of parental and iPSC pairs have demonstrated that there are, on average, 6-12 coding SNVs per iPSC line (1, 7-9) with varying theories regarding when these SNVs arose in the iPSCs. Most reports attributed de novo SNVs or CNVs in iPSCs to reprogramming-induced stress or long-term in vitro culture (2,3,7,8,(10)(11)(12)(13)(14). However, several studies also suggested that many of the "de novo" variants were either inherited rare preexisting variations in the founder source cells or benign variants regardless of reprogramming method used (1,9,15,16).To determine whether iPSCs are inherently more likely to accumulate mutations and further elucidate the origin of genomic variants present in iPSCs, we have undertaken a unique approach by establishing clonal fibroblast subclones and iPSCs from the same fibroblast population to directly assess whether the reprogramming process leads to more mutations by next-generation sequencing. De novo mutations, which were not detected in the starting pooled parental fibroblasts, were identified in each daughter cell line, both fibroblast subclones and iPSCs. These putative de novo mutation sites were then deep sequenced in the parental fibroblast population to determine whether they were present at low frequency as mosaic variants. In addition, highresolution single nucleotide polymorphism (SNP) arrays were used to search for de novo CNVs in both fibrobla...

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“…Thus, those plots farthest from the diagonal are for the cells with the poorest whole genome coverage. We follow Szulwach et al 35 and calculate G, the Gini coefficient for the Lorenz plots, to quantify the uniformity of the genome coverage. The Gini coefficient G is calculated as:…”

Section: Coveragementioning

confidence: 99%

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

et al. 2018

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Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

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Single-Cell Genetic Analysis Using Automated Microfluidics to Resolve Somatic Mosaicism

Cited by 42 publications

References 32 publications

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

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